Recombinant adeno-associated virus (AAV) is a highly promising gene therapy vector for numerous reasons, including its non-pathogenicity and its ability to induce long-term expression of a transgene in multiple target cell types. However, the large scale production of AAV is complex, either involving transient plasmid transfection or co-infection with a helper virus (such as adenovirus), which must eventually be removed from the product to avoid helper-induced pathogenicity. In addition, methods of generating recombinant AAV (rAAV) involving either helper plasmid or helper virus result in a mixture of encapsidated rAAV and AAV particles that lack viral nucleic acid (e.g., “empty shells”). Current methods for removing the empty shells include ultracentrifugation through a cesium chloride (CsCl) gradient. However, CsCl is toxic to mammals, and the CsCl gradient/ultracentrifugation method is not amenable to larger scale preparation. Furthermore, the cesium can inactivate viral particles that include viral genomes.
There is a need in the art for methods for selectively inactivating empty viral particles while leaving viral particles that contain viral genomes that are active and intact.
Literature
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